Membrane anchoring via glycosylphosphatidylinositol (GPI) structures is a ubiquitous mechanism whereby many functionally diverse proteins are naturally linked to cell surfaces, and by means of which any protein of interest can be experimentally attached to cells. From previous work by several labs including our own, methods for GPI reanchoring of any protein and incorporating the modified protein into cells are developed and the reactions that are involved in intracellular GPI assembly are characterized. In recent studies, we have been exploiting novel GPI- anchoring mutant lines (designated K and 0.48) that fail to donate terminal GPI precursors and deacylate the donated precursors to examine the final GPI transfer steps in which preassembled anchors are appended to nascent polypeptides. Additionally, we have been utilizing GPI- reanchored proteins for engineering tumor and other cell surfaces, and have initiated investigations into unexpected reports that GPI-anchored proteins may possess an ability to transfer between cells in vivo. The objectives of our current proposal are to build upon this work in order to optimize methods for resurfacing cancer and other cells with immunomodulatory factors and gain insights into the biological properties of incorporated proteins in the in vivo setting. Specifically our objectives are to 1) further characterize the properties of GPI- anchored proteins following their incorporation into nucleated cells, 2) utilize an in vitro translation system in conjunction with our GPI mutant cells to investigate the effects of modulating GPI anchor transfer, 3) exploit the mammalian translation system to investigate how GPI production and attachment to different proteins are regulated physiologically, and 4) employ knock-out mice deficient in GPI-anchored proteins and transgenic mice with GPI-anchored reporters to study whether intercellular transfer occurs physiologically and if so, analyze the properties of the in vivo transferred proteins. The data obtained should not only have relevance for cell surface engineering with GPI- anchored proteins for various applications, but also contribute to our understanding of GPI-anchored protein physiology in vivo.